Delivery of granulocyte-macrophage colony-stimulating factor in bioadhesive hydrogel stimulates migration of dendritic cells in models of human papillomavirus-associated (pre)neoplastic epithelial lesions

Abstract

Because of the central role of dendritic cells and/or Langerhans cells(DC/LC) in the induction of cellular immune responses, pharmacological agents that modulate the recruitment of these cells might have a clinical interest. The present study was designed to evaluate the capacity of several pharmaceutical formulations to topically deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) on human papillomavirus (HPV)-associated genital (pre)neoplastic lesions. The formulations were evaluated for their bioactivity and for their potential to recruit DC in organotypic cultures of HPV-transformed keratinocytes. We found that a bioadhesive polycarbophil gel (Noveon) at pH 5.5 is able to maintain the bioactivity of GM-CSF at 4 or 37 degrees C for at least 7 days, whereas a decreased activity of GM-CSF was observed when the molecule is included in other polymer gels. GM-CSF incorporated in the polycarbophil gel was also a potent factor in enhancing the colonization of DC into organotypic cultures of HPV-transformed keratinocytes since the infiltration of DC in the in vitro-formed (pre)neoplastic epithelium was very low under basal conditions and dramatically increased in the presence of GM-CSF gel. We next demonstrated that GM-CSF incorporated in polycarbophil gel induces the recruitment of human DC in a human (pre)neoplastic epithelium grafted into NOD/SCID mice. The efficacy of GM-CSF in this formulation was equivalent to that observed with liquid GM-CSF. These results suggest that GM-CSF incorporated in polycarbophil gel could play an important role in the recruitment of DC/LC in mucosal surfaces and be useful as a new immunotherapeutic approach for genital HPV-associated (pre)neoplastic lesions.

FIG. 1.

Proliferation response of TF-1 cell line after incubation with GM-CSF extracted at different time intervals from the hydrogels stored at 4 or 37°C. Growth was measured by ³H-labeled thymidine incorporation. Each point shows the mean of six replicates, and bars represent standard errors.

FIG. 2.

Double staining and flow cytometry analysis of DC generated from cultures of adherent human PBMCs in the presence of GM-CSF and IL-4 during 7 days. Results are displayed as dot plots. Cells were labeled with CD1a-fluorescein isothiocyanate antibody versus indicated phycoerythrin-conjugated antibodies. Quadrant setting was performed according to the reactivities of isotype-matched controls. One representative experiment is shown out of more than 10 experiments performed.

FIG. 3.

Quantitative evaluation of DC infiltration into organotypic cultures of HPV-transformed keratinocytes under the influence of GM-CSF incorporated or not into polycarbophil gel. The penetration of DC was followed by immunolabeling with anti-CD1a. Results are expressed as the mean percentages of surface labeled with anti-CD1a compared to unlabeled surface of the epithelial sheet ± the standard deviation (five experiments). Asterisks indicate statistically significant differences (***, P < 0.001).

FIG. 4.

Quantitative evaluation of DC infiltration into transplants of HPV-transformed keratinocytes. The results are expressed as numbers of DC per square millimeter of HPV-positive epithelium ± the standard deviation (five experiments were conducted for each condition). Asterisks indicate statistically significant differences (***, P < 0.001).